A while ago, I talked about the influence of vaccination on Marek’s Disease Virus. Marek’s Disease Virus (MDV) is a ubiquitous chicken virus that causes tumors in infected birds. It’s a big problem for commercial flocks, and so vaccines against MDV are widely used. MDV is a herpesvirus, and in general it’s been hard to come up with really effective vaccines against herpesviruses. MDV is no exception; the vaccine prevents disease, but doesn’t prevent infection very well. As a result, there’s apparently been selection pressure on MDV to become more virulent over time.

There are quite a few vaccines against herpesvirus diseases, but most of them are in domestic animals rather than humans (and most of them would no be acceptable in humans, because of their lack of efficacy or their risk). In spite of a lot of research, there’s really only one human anti-herpesvirus vaccine, the chicken-pox vaccine. (There are eight known human herpesviruses, if you’re keeping count. Chicken-pox is caused by the varicella-zoster virus, VZV.) The MDV observation has left me wondering if the chicken-pox vaccine might lead to evolution of VZV virulence.

A recent paper1 looks at a related issue: Evolution of the vaccine virus itself. Before talking about it, I should note that this is mainly a hypothetical concern; the VZV vaccine has been used widely for a long time, and has been clearly shown to be extremely safe and quite effective; whereas the actual disease — though usually fairly mild — used to hospitalize about 10,000 people per year (mostly children) and kill around a hundred people per year, in the USA alone.

The “vaccine strain” of VZV (the “vOka virus”) is not actually a single, pure, strain of virus. It’s a mixture of strains, each with a different set of changes, and with more or less similarity to wild-type virus. It’s also known that the vaccine occasionally causes a rash in vaccinees, although this disease is much milder than the natural infection.

So the question that Quinlivan et al1 asked was, What’s actually causing the rash? Is it a vaccine strain that’s relatively close to the wild-type virus? Is it a more variant strain? Is it caused by the same mix of strains as found in the original vaccine? Or is it something that wasn’t originally present in the vaccine — a revertant strain, that’s evolving back to become a virulent wild strain again?

It turns out that the rashes are not associated with new mutations: The virus is not reverting to wild-type or developing new virulence mechanisms. On the other hand, viruses associated with rashes are not identical to the original vaccine mixture. Instead, a particular subset of the vaccine strains seems to be better at causing rash:

The genotypes that carry one or more of the four selected mutations have outcompeted other components of the live vaccine inoculum. … the development of rash after vOka vaccination is due to selection of viral strains that occur infrequently in the vaccine and probably depends on specific interactions by these viruses with host immunity.

What can we learn from this? First, this may offer an opportunity to improve the vaccine. If the more virulent (rash-causing) variants can be removed from the vaccine strain, then presumably the vaccine will be safer. (We don’t know, though, whether these more virulent strains are also disproportionately responsible for immunity.) Second, the authors propose a mechanism that may make a strain more virulent — the ability to avoid T cell responses — and suggest that there may be a particular subgroup of people at greater risk of rash — those who don’t have a particular T cell recognition motif:

One corollary of this interpretation is the prediction that the vaccine virus will be found to spread more effectively in subjects who are HLA A2-negative or have lost immunity to this epitope.

More generally, this information may also be useful in understanding how viruses like VZV interact with the host immune response over long periods.

To identify hitherto-unknown rodent-associated herpesviruses, we captured M. musculus, R. norvegicus, and 14 other rodent species in several locations in Germany, the United Kingdom, and Thailand. … we detected 17 novel betaherpesviruses and 21 novel gammaherpesviruses but no alphaherpesvirus. … it is also possible that rodent alphaherpesviruses either never developed or became extinct earlier during herpesvirus evolution.

Comment: Herpesviruses are an ancient, ubiquitous family of viruses that as a group infect essentially every species of mammal, and probably virtually every species of bird and reptile as well.

In mammals, herpesviruses are divided into three families (alpha, beta, and gamma-herpesviruses) that are distinct in their biological activities. But the ancestral herpesviruses seem to have been alpha-herpesviruses; herpesviruses of birds and reptiles are all alpha. Presumably the beta- and gamma families arose from the ancestral alphas, some time after mammals diverged from their own, reptilian, ancestors.

It’s puzzling, then, that mice and rats — which are, as this quote shows, infected with dozens of different herpesviruses — don’t appear to have any natural alpha-herpesvirus infections. Why? I have no idea. Could there be some intrinsic incompatibility between the alpha-herpesvirus lifestyle and rodents? Or could there have been some strange bottleneck that caused rodent herpesviruses to go extinct tens of millions of years ago?

Making things even more puzzling is the fact that rabbits apparently do have at least one alpha-herpesvirus. One was just isolated from domestic rabbits in Alaska (of all places).1 We know almost nothing about this virus yet, so it’s possible that rabbits aren’t the natural host; but if they are, then the rodent lineage presumably lost their complement of alpha-herpesviruses somewhere after the rabbit lineage branched off.2

Antibodies bind to their target antigens because the bumps and crannies in an antibody’s binding site complement the crannies and bumps in the antigen.

Antibodies were invented by jawed vertebrates; sharks and all their progeny have antibodies, while lampreys and hagfish, which have a common ancestor with sharks some 500 million years ago, don’t use antibodies as such. Lampreys do, however, have circulating proteins that apparently play the same role as do antibodies, binding with high avidity to antigen and offering protection against pathogens. Those circulating protein (variable lymphocyte receptors; VLRs) look very different from antibodies, and generate diversity in a very different way. I’ve talked about these things before, here and here.

VLRs are interesting because (among other things) they combine extreme specificity and high binding affinity with great stability, making them intriguing pharmaceutical agents.

Now, Ian Wilson’s group has determined the structure of a lamprey VLR in combination with its bound antigen1 (in this case, a blood-group sugar, H-trisaccharide). Here it is, on the left. For comparison (but not to scale), a mouse antibody (in blue, as ribbons) bound to its antigen (in red, showing its surface) is on the right. (Click for larger versions.) The VLR seems to bind at the “bottom” of its concave face, quite different from the antibody, as you’d expect.

Our findings illustrate that increasing genomic complexity of the Mup gene family is not evolutionarily isolated, but is instead a recurring mechanism of generating coding diversity consistent with a species-specific function in mammals.

The 1918 influenza pandemic that killed between 20 million and 100 million people world-wide was unusual in a lot of ways. One of the most extraordinary things about it was not just the high mortality rate, but the mortality pattern. Normally influenza kills the very old and the very young; but the 1918 flu killed young adults as well — people in the prime of life, who normally are highly resistant to death from influenza. The famous graph below1 (click for a larger version) shows this; the dashed line is mortality vs. age for “normal” influenza outbreaks (the “U-shaped curve”), and the solid line shows mortality vs. age for the 1918 flu (a “W-shaped curve”).

There are two ways to look at the W-shaped curve. You can either ask, Why did the young adults die? Or you can ask, Why did the older adults NOT die? In other words, was there something that protected the older adults that the younger ones didn’t have?

One explanation (and I can’t find the original paper to give credit) is that somewhere around 1870-ish (that is, 45 or 50 years before 1918) some other influenza strain infected the population, and gave a little bit of cross-protection against the 1918 strain. People alive in 1870 were exposed to this (hypothetical) strain of virus, developed immunity, and fifty years later were protected against the 1918 strain.2 Of course, supporting this idea is the recent paper3 that received a fair bit of attention, showing that survivors of the 1918 pandemic still have specific immune responses to that virus, 90 years later — so certainly immunity could last fifty years.

One of the problems with influenza vaccination, of course, is that the virus changes. New strains arise and mutate, and the vaccines have to match the major circulating virus pretty well. Antibodies against one major strain of influenza don’t do a good job against a shifted strain. We see this is the paper I just mentioned — hardly anyone younger than 90 responded to the 1918 flu, even though they had been exposed to influenza viruses (of different types) every year for up to 80 years.

It would be nice if there was a vaccine that gave resistance to many strains of influenza, and we didn’t have to develop new vaccines ad hoc each year, based on imperfect guesses as to which viruses will circulate six months in the future. What’s more, we don’t really have good vaccine strains for novel influenza strains — like, say, whichever strain of avian influenza eventually succeeds in making the jump to humans.

Our present vaccines against flu are designed to raise protective antibodies. Antibodies in general target the outside of the viruses, which are intrinsically variable; hence the need to customize the vaccine strain with the circulating strain. Cytotoxic T lymphocytes, on the other hand, at least have the potential to target internal components of the viruses, which may have different constraints and therefore not be able to change as much. Could these CTL be more cross-reactive than antibodies?

It turns out that, yes, CTL are quite cross-reactive. A paper from Tao Dong’s and Sarah Rowland-Jones’ lab4 looked at anti-influenza T cells from normal volunteers (who had, of course, been exposed to the usual influenza A strains that sweep around the world each year).

Memory CD4+ and CD8+ T cells isolated from the majority of participants exhibited human influenza-specific responses and showed cross-recognition of at least one H5N1 internal protein.

The bad news is that these cross-reactive T cells are not very effective even against conventional influenza strains. Most of us are walking around with cross-reactive T cells, yet we still need antibody (such as from the vaccine) to actually be resistant to infection with one of the cross-reactive viruses. The good news is that the T cells aren’t entirely useless. It’s been shown several times that they do offer some protection, and in fact may be one reason that healthy adults are relatively resistant to influenza (the U-shaped curve above) — the very young would not have been previously exposed so wouldn’t have any cross-reactive T cells, while the very old often have defects in T cell responses. As Peter Doherty notes in his accompanying commentary:

The argument is that if seasonal influenza infection does promote cross-reactive T cell responses, then why do so many people get sick every one or two years? The counter-argument is, of course, that the majority of individuals may be protected from more serious disease by their T cell response.5

If the T cells that are normally present are partially protective, could they be cranked up to offer more protection? For example, what if vaccines (as well as triggering antibody responses) also tried to boost these cross-reactive memory responses? Potentially, this could lead to people who have long-term resistance to many influenza viruses, avoiding the need for annual re-vaccination, as well as having immunity to new influenza viruses that aren’t otherwise covered by the vaccinations; such as avian influenza.

Most influenza vaccines used today are killed (inactivated) vaccines, which are not very good at inducing CTL responses. However, cold adapted live attenuated influenza vaccines (LAIVs) are also used in some cases, and these should act as boosters for CTL:

… it would be worth evaluating the extent of cross-protection against H5N1 potentially conferred by currently available seasonal human LAIVs. Memory T cell populations boosted by these vaccines may in theory cross-react and provide partial protection against H5N1 by targeting highly conserved internal virus proteins. … The aim of such T cell-based approaches would be to provide broader partial protection against overwhelming infection and help lower morbidity and mortality rather than to provide complete protection against establishment of infection. This would be a highly relevant and perhaps more realistic public health goal in a pandemic situation.4

There were influenza pandemics in 1830-33; 1889-1890; and 1900; as well as epidemics in some other years. That was long before influenza virus was identified, and we have no idea what those strains were.[↩]

XPlasMap 0.99 is out; download it here. A partial list of new features and changes:

New features

Auto-positioning for enzymes and gene text

Import from EMBOSS

Ovals, rectangles, and arrows for annotations

Convert restriction sites to MCS/MCS to restriction sites

Change feature fonts on an individual basis

Insert, copy, and cut fragments by restriction site

Export to plain text

Import circular DNA from GenBank correctly

Plasmid name and description are moveable

Changes

Determine PNG and JPG resolution at export

Checkbox to identify ORFs in Genbank during import

Change “Insert fragment” shortcut from “?I” to “?V”

Change “Edit plasmid info” shortcut to “?I”

“Info” icon in toolbar to edit plasmid info

Many bugfixes

If you downloaded version 0.99 when I mentioned it earlier here, please get the new version anyway; I made a couple minor bugfixes in the meantime.

I’ve tested it on several computers, including Intel and PowerPC, MacOS4 and MacOS5, but there are some combinations of those I haven’t been able to test. Hopefully there are no configuration-dependent bugs.

The other day I talked about Jim Allison’s paper1 that proposed (among other things) that irradiation of tumors might help the immune system detect them. A quite unrelated paper2 seems to have almost accidentally helped test that suggestion.

Allison’s group was looking at the puzzling fact that circulating anti-tumor T cells don’t seem to correlate well with effective anti-tumor immune responses. They suggest that part of the problem is that the blood vessels that supply tumors are often abnormal, and these altered blood vessels don’t allow T cells to enter the tissues properly. However, irradiating those blood vessels made them closer to normal, allowed the T cells to enter the tumor more effectively, and led to more complete immune clearance of the tumor.

The new paper I mentioned2 wasn’t specifically looking at tumor vasculature at all. They were working with a model of brain cancer (a particularly difficult nut to crack, immunologically). They used transgenic mice, that express a tumor antigen (the SV40 virus large T antigen) in some brain cells; these mice develop brain cancer and die by 3 or 4 months of age. They’re immunologically tolerant to the tumor antigen, as you’d expect; but transferring tumor antigen-specific T cells from non-transgenic mice helped them survive longer. In this particular paper, they try to narrow down the specific conditions that allow immune rejection of the tumor.

They found a number of important factors, but the interesting one in light of the Allison paper was that “successful adoptive immunotherapy of T Ag-induced tumors still required prior conditioning of the host with gamma irradiation”. Just transferring the T cells helped a bit, delaying death by a couple of months; but irradiating the tumors and transfering the T cells completely eliminated the tumors. They didn’t specifically look at the tumor blood vessels (the Allison paper wasn’t out when this work was done, of course), but they did note that irradiation correlated with increased T cell accumulation in the brain.

Another interesting point is that the irradiated tumors were cleared even when only a single T cell epitope was targeted. That’s an important finding, because one might expect that the tumor might be able to recur because of immune escape. (That is, tumor cells with fortuitous mutations in that epitope would not be killed by the T cells, and would be able to regrow and recur.) In fact, that might have happened with the non-irradiated mice, in which the tumor shrank briefly then rapidly recurred. (They didn’t check the epitope, so it’s equally possible that something like tolerance or deletion of the T cells was involved in this situation.) Still, it seems that, at least in this model, you can get rid of a tumor altogether, even if you only have a single tumor epitope to work with. Given the limited number of tumor epitopes we have to work with, that’s pretty encouraging.

Finally managed to get XPlasMap 0.99 running on MacOS10.4/PowerPC, as well as MacOC10.5/Intel. My wife went out to the movies, so after I had popcorn with the kids, and played Scrabble Junior, and a game of chess, and discussed the Dropkick Murphys and spiders and batman and Jon Papelbon and other essential snippets of 21st-century wisdom, and put the kids to bed; after that I was able to get on to the Powerbook and (interrupted by three emails with data from my grad student, who was feeling justifiably triumphant after a very productive week) re-install all the appropriate modules (including a minor-release downgrade of wxPython, since the latest has a weird bug I didn’t want to track down) — and with a bit of tweaking and adjustment, I think XPlasMap is now running more or less correctly on both platforms.

I will probably announce it some time next week. If you want a sneak preview, try here.

Emerging diseases don’t arise from nothing; they are established diseases that move away from their previous niche. Usually when a pathogen tries to move out of its comfort zone, it drives down a dead end; it might infect a few individuals, but it doesn’t spread effectively, and it fizzles out.

Of course, we don’t usually hear about the fizzles; we hear about the successes (HIV) and the partial successes (SARS, avian influenza, Sin Nombre virus, and many more). Even so, many people don’t seem to know much about one of the most spectacularly successful emerging diseases of the 20th century.

In 1978, dogs around the world suddenly began to die, developing a bloody diarrhea and rapidly (often overnight) progressing to fatal dehydration. This turned out to be due to canine parvovirus 2 (CPV2); dogs were already known to have a parvovirus (CPV1) but this one was new, and lethal. It exploded around the world within a few months, infecting millions and killing thousands of dogs.

The good news was that this virus turns out to be effectively controlled by the immune system. Its initial explosion was through a naïve ecosystem, dogs that had never been exposed to anything like this virus and that had no immunity to it. A single exposure to the virus gave long-lasting immunity, and this immunity could be passed to pups by nursing. Along with the rapid development of a new vaccine, the worldwide dog population rapidly become, by and large, resistant to CPV2. The virus is still with us, but because of herd immunity usually isn’t a cause of epidemics any more. 1

Canine parvoviruses is one of the very few, if not the only, example of a pandemic species-switching virus in the modern age of molecular biology. We’ve been able to watch the virus mutating, spreading, and responding to the environment almost from the outset. (We don’t know exactly when the virus entered the dog population; it must have been before 1978, perhaps well before, and circulated in a small pool of animals for a while before its sudden eruption. These initial stages would be very interesting to analyze as well, to see what, if anything, allowed the virus to switch from cycling through a small population to infecting everything.)

The actual origin of CPV2 is a little controversial. The original suggestion was that it arose from a feline parvovirus, feline panleukopaenia virus (FPV). In fact, it was believed for a while that it might have arisen from contamination of canine vaccines, by the feline virus — which would help explain the almost simultaneous appearance worldwide. But now it’s agreed that CPV2 could have arisen from a parvovirus of some other carnivore — mink, or perhaps foxes or raccoons — instead of from FPV, and almost certainly wasn’t from a vaccine contaminant. (FPV still looks like the most likely source, though.)

The actual mutations in CPV2 that allowed it to spread into dogs have been pinpointed — there are changes that allow the virus to infect canine cells, which FPV can’t do. Not surprisingly, though, CPV2 has continued to mutate rapidly since 1978. 2 It’s moved into a new ecosystem, and it’s adapting to that ecosystem in a hurry. New variants of CPV2 are now showing the ability to infect cats (again?); something to watch for.

Viruses that successfully switch hosts are rare, but potentially catastrophic, events. The lessons of CPV might help us understand how, say, influenza viruses might shift from chickens to humans (and this hasn’t gone unnoticed;3 I’m not the first to make the connection by any means). CPV is a unique resource, because we have examples of so many of the steps in the evolution of a virus that’s adapting to a new host.

An excellent review of the disease itself is in: Carmichael LE (2005) An annotated historical account of canine parvovirus. J Vet Med B Infect Dis Vet Public Health 52:303-311. However, I think he’s overconfident, or outdated by now, in his assertion that the virus definitely didn’t arise from feline panleukopaenia virus.[↩]

… adaptation of H9 viruses to land-based birds can lead to strains with expanded host range…. some bird species may act as a bridge in the generation of strains that can replicate more efficiently in mammals. In our particular case, quail provided an environment that improved the ability of a duck H9N2 virus to replicate in mice.